PROJECT SUMMARY Neonatal stroke is an important cause of death and disability, resulting from multiple pathways of cell death and dysfunction that evolve over a prolonged period of time. There is insufficient knowledge regarding the role of angiogenesis in the response to focal ischemia-reperfusion injury that is the most common cause of early stroke. Angiogenesis and neuronal repopulation occur in close proximity, with paracrine factors supporting neuron-endothelial cell interactions that are critical for repair. Modulating this neurovascular niche may be a potential target for enhancing outcomes after ischemic brain injury in the newborn period. Cell-based therapies have emerged as a promising treatment for CNS disease, although the mechanism of repair has been controversial. Mesenchymal stem cells (MSC) play a role in vascular formation and secrete pro-angiogenic factors, express a number of growth factor receptors, and may preferentially differentiate into cells capable of building new blood vessels after injury. MSC treatment has been shown to improve histological and functional outcomes after ischemic injury, even when therapy was delayed, but it is only partially reparative. Modifying MSC in a way that enhances effects on the neurovascular unit may provide additional benefit. Our overall objective is to determine the mechanism of regeneration and repair with delayed cellular therapy for neonatal stroke by focusing on the vascular response in the injured brain. In Aim 1, we will test the hypothesis that MSC pre-exposed to EPO will enhance angiogenesis and vascular remodeling following neonatal focal ischemia-reperfusion injury in the rat more than MSC or EPO therapy alone. In Aim 2, we will clarify to what extent this modified cellular therapy modulates long-term repair by using a number of techniques to quantify cell fate, gross histology and long-term sensorimotor and cognitive outcomes. Finally, in Aim 3, we will define the roles of specific downstream signaling pathways on angiogenesis and repair following ischemia- reperfusion injury and cellular therapy. This will determine critical, modifiable pathways important for angiogenesis in the developing brain and following focal injury, which can be further studied to enhance long- term repair. Our primary hypothesis is that delayed treatment with MSC pretreated with EPO will promote vascular growth and remodeling, increase neurogenesis, and improve long-term histological and functional outcomes after neonatal stroke. This will provide a late treatment option for a common cause of early brain injury, where diagnosis is often delayed.